Glycans on proteins are classified as N-glycans and O-glycans. In N-glycans, an amido group in a side chain of asparagine (N) is N-glycosylated. In O-glycans, an alcohol in a side chain of serine (S) or threonine (T) is glycosylated. Glycoproteomics places importance on determination of types of glycans (structures), and types and glycosylation sites of proteins. Glycosylated proteins can be converted into glycosylated peptides by enzymatic digestion such as tryptic digestion. Thus, they can be identified if the amino-acid sequences of glycosylated peptides can be analyzed and determined. In practice, however, glycans have complicated structures, and this inhibits satisfactory analysis of glycosylated peptides.
N-glycosylated peptides have been analyzed by a technique of liberating a glycosylation site from the peptide moiety through enzymatic digestion. It is difficult, however, to determine the types and glycosylation sites of the original peptides and proteins according to this technique, although constitutive glycans can be analyzed. As another possible solution, a technique has been proposed in which the glycosylation site of a peptide is labeled with an isotope upon separation of a glycan from the peptide through enzymatic digestion. It is difficult, however, to determine the types of glycans including structural isomers according to this technique, although the types and glycosylation sites of proteins can be determined. In analyses of O-glycosylated peptides, there has been proposed a technique of chemically derivatizing glycans. This technique, however, is independent of peptide analyses, and the acquired data are not satisfactorily tied with the peptide analyses (J. Zhang, L. L. Lindsay, J. L. Hedrick. C. B. Lebrilla, Strategy for profiling structure elucidation of mucin-type oligosaccharides by mass spectrometry, Analytical Chemistry Vol. 76 (2004) 5990-6001). Thus, different techniques have been applied to analyze N-glycans and to analyze O-glycans, respectively.
In contrast, attempts have been made to directly analyze glycosylated proteins and peptides by mass spectrometry without liberating glycans therefrom. JP-A No. 2005-300420, for example, discloses a technique for analyzing a glycosylated peptide by carrying out tandem mass spectrometry of protonated or cationized ions derived from the glycosylated peptide through collision-induced dissociation (CID) using a mass spectrometer. When an ion derived from the principal chain of a peptide and another ion derived from a glycan are separately and independently detected, these ions are further subjected to tandem mass spectrometry (MS/MS/MS), respectively, according to this technique. Thus, information on detected ions is simplified. In addition, there have been proposed a technique for analyzing a glycosylated peptide by subjecting a positive ion derived from the glycosylated protein to CID in combination with electron capture dissociation (ECD), infrared multi-photon dissociation (IRMPD), and/or electron transfer dissociation (ETD). This technique has been proposed by K. Hakansson, H. J. Cooper, M. R. Emmett, C. E. Costello, A. G. Marshall, C. L. Nilsson in Electron capture dissociation and infrared multiphoton dissociation MS/MS of an N-glycosylated tryptic peptide to yield complementary sequence information, Analytical Chemistry Vol. 73 (2001) 4530-4536, and by J. M. Hogan, S. J. Pitteri, P. A. Chrisman, C. A. McLuckey in Complementary structural information from a tryptic N-linked glycopeptide via electron transfer ion/ion reactions and collision-induced-dissociation, Journal of Proteome Research Vol. 4 (2005) 628-632. In addition, there has been proposed a technique for analyzing a glycosylated peptide by carrying out positive-ion CID tandem mass spectrometry in combination with negative-ion CID tandem mass spectrometry by K. Deguchi, H. Ito, Y. Takegawa, N. Shinji, H. Nakagawa, S. Nishimura in Complementary structural information of positive- and negative-ion MSn spectra of glycopeptides with neutral and sialylated N-glycans, Rapid Communications in Mass Spectrometry Vol. 20 (2006) 741-446.